Compositions containing prodrugs of florfenicol and methods of use

ABSTRACT

The present invention provides compositions and methods for administering florfenicol to mammals. The compositions contain a prodrug of florfenicol in a pharmaceutically acceptable carrier. In one embodiment the prodrug is an esterized form of florfenicol. Examples of suitable prodrugs include one or a combination of one or a combination of the following: florfenicol acetate, florfenicol propionate, florfenicol butyrate, florfenicol pentanoate, florfenicol hexanoate, florfenicol heptanoate, florfenicol octanoate, florfenicol nanoate, florfenicol decanoate, florfenicol undecanoate, florfenicol dodecanoate, and florfenicol phthalate. In another embodiment the prodrug is converted into the florfenicol in vivo by the action of one or more endogenous esterases. The invention also provides new compounds, pharmaceutical compositions containing the compounds, and methods for their administration.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.10/623,114, filed Jul. 18, 2003, now U.S. Pat. No. 7,439,268.

FIELD OF THE INVENTION

The present invention pertains to prodrugs for use in mammals.

BACKGROUND OF THE INVENTION

The following discussion of the background of the invention is merelyprovided to aid the reader in understanding the invention and is notadmitted to describe or constitute prior art to the present invention.

Florfenicol is a structural analog of chloramphenicol and thiamphenicol,having a fluorine atom instead of the hydroxyl group located at C-3 inthe structure of chloramphenicol and thiamphenicol. Bacterial resistanceto chloramphenicol and thiamphenicol is usually attributed toacetylation of the 3′ hydroxyl group by the action of chloramphenicolacetyl transferase. Since florfenicol does not possess the 3′ hydroxylgroup such a mechanism of inactivation is not possible with florfenicol,thus making florfenicol a highly useful antibiotic.

Florfenicol is active against a variety of microorganisms includingCitrobacter, Proteus mirabilis, Proteus sp., Shigella, Salmonella,Providencia, Bacteroides, Staphylococcus aureus, Enterococci,Pasteurella haemolytica, Pasteurella multocida, Haemophilus somnus, andHaemophilus influenza. Conversely, Serratia marcescens, Pseudomonasaeruginosa, and Acinetobacter, have been found to be resistant toflorfenicol. Literature suggests that typical MICs (minimum inhibitoryconcentrations) of florfenicol for various bacteria range from 0.3-1μg/ml.

Dosage schedules of antibiotics are designed to maintain serum or tissuelevels above the MIC for the target organisms for a period of timesufficient to clear the infection. Drugs that are cleared rapidly mustbe administered more often to maintain effective levels. In the case ofsome highly hydrophilic drugs, the drugs are cleared from the system sorapidly that their use requires multiple daily doses. Therefore, thereis a need for increasing the effectiveness of known hydrophilicantibacterial compounds, such as florfenicol.

SUMMARY OF THE INVENTION

In one aspect, the present invention provides compositions containing aprodrug of florfenicol and a pharmaceutically acceptable carrier in aninjectable composition. In one embodiment the prodrug of florfenicol ispresent in the composition at a concentration of at least 200 mg/ml,while in another embodiment the prodrug of florfenicol is present in thecomposition at a concentration of about 300 mg/ml. In still anotherembodiment the prodrug is an esterized form of florfenicol. In variousembodiments the prodrug is one or a combination of the following:florfenicol acetate, florfenicol propionate, florfenicol butyrate,florfenicol pentanoate, florfenicol hexanoate, florfenicol heptanoate,florfenicol octanoate, florfenicol nanoate, florfenicol decanoate,florfenicol undecanoate, florfenicol dodecanoate, and florfenicolphthalate. In one embodiment the prodrug is converted into theflorfenicol in vivo by the action of an esterase.

By “prodrug” is meant a chemical derivative compound that is transformedin vitro to yield florfenicol through the action of one or moremammalian enzymes. In one embodiment the mammalian enzyme is anesterase. In various embodiments prodrugs are derivatized through anester linkage between a hydroxyl group of the drug molecule and thecarboxyl group of an external acid, or vice versa, to form an esterizedform of florfenicol. The term “prodrug ester group” refers to any ofmany ester-forming groups that are hydrolyzed in the mammalian body.Examples of prodrug ester groups and external acids include acetyl,propionyl, butyryl, pentanyl, hexanyl, heptanyl, octanyl, nonyl,decanyl, undecanyl, dodecanyl, and phthalyl. Other examples of suitableprodrug ester groups and external acids can be found in the book“Pro-drugs as Novel Delivery Systems,” by Higuchi and Stella, Vol. 14 ofthe American Chemical Society Symposium Series, American ChemicalSociety (1975).

By a “pharmaceutically acceptable carrier” is meant a non-toxic, inert,solid, semi-solid, or liquid filler, diluent, encapsulating material, orformulation auxiliary of any type. In one embodiment thepharmaceutically acceptable carrier is propylene glycol. But many othercarriers are available. Examples of pharmaceutically acceptable carriersinclude, but are not limited to, pyrrolidone, N-methylpyrrolidone,polyethylene glycol, propylene glycol, glycerol formal, isosorbiddimethyl ether, ethanol, dimethyl sulfoxide, tetrahydrofurfuryl alcohol,triacetin, or any combination of these, or another solvent found to havesimilar acceptable properties such as being non-toxic and soluble inwater. In one embodiment the carrier is propylene glycol in glycerolformal (e.g., in 10% glycerol formal). Other pharmaceutically acceptablecarriers include sugars such as lactose, glucose, and sucrose; starchessuch as corn starch and potato starch; cellulose and its derivativessuch as sodium carboxymethyl cellulose, ethyl cellulose and celluloseacetate; powdered tragacanth; malt; gelatin; talc; excipients such ascocoa butter and suppository waxes; oils such as peanut oil, cottonseedoil, safflower oil, arachis oil, sesame oil, olive oil, corn oil,soybean oil, and saw flower oil; glycols, such as propylene glycol;polyols such as glycerin, sorbitol, mannitol and polyethylene glycol;esters such as ethyl oleate, isopropyl myristate, and ethyl laurate;agar; ethyl alcohol; as well as other non-toxic compatible substancesused in pharmaceutical formulations. Any of these or mixtures thereofcan be prepared in a liquid form or dissolved in a suitable liquid toform the carrier. Still other pharmaceutically acceptable carriersinclude water insoluble or water immiscible solvents. Examples of theseinclude, but are not limited to, isopropyl myristate, and ethyl lactate.

By “injectable” is meant a composition or formulation that is suitablefor placing into a syringe and injecting into the mammalian body. Thecomposition and vehicle are tissue-compatible and should not rely onconstituents that are likely to elicit an allergic response. Injectablecompositions can be injected into the mammalian body without causingadverse effects due to the presence of solid materials in thecomposition. Solid materials include, but are not limited to, particles,crystals, a gummy mass, and a gel. The injectable compositions can beinjected intra-muscularly, intra-venously, sub-cutaneously,intra-dermally, intra-peritoneally, or parenterally, or other suitablemodes of injection into the mammalian body.

One injectable composition of the invention contains one or morecompounds of the present invention dissolved in glycerol formal with 10%propylene glycol. In one embodiment the composition contains asuspension or solution of one or more compounds of the present inventiondissolved in an oil, for example arachis oil or sesame oil. Thecompositions can also contain one or more compounds of the presentinvention dissolved in a solvent such as pyrrolidone,N-methylpyrrolidone, polyethylene glycol, propylene glycol, glycerolformal, isosorbid dimethyl ether, ethanol, dimethyl sulfoxide,tetrahydrofurfuryl alcohol, triacetin, or any combination of these, oranother solvent found to have similar acceptable properties such asbeing non-toxic and soluble in water. The compositions can optionallycontain a chelating agent, for example ethylenediamine tetracetic acidand an anti-oxidant, for example sodium metabisulphite.

In another aspect the present invention provides novel compounds. Thecompounds are one or more compounds having a structure selected from thefollowing: florfenicol acetate, florfenicol propionate, florfenicolbutyrate, florfenicol pentanoate, florfenicol hexanoate, florfenicolheptanoate, florfenicol octanoate, florfenicol nanoate, florfenicoldecanoate, florfenicol undecanoate, florfenicol dodecanoate, andflorfenicol phthalate.

In another aspect the present invention provides pharmaceuticalcompositions for administration to a mammal. The pharmaceuticalcompositions contain one or more compounds described herein and apharmaceutically acceptable carrier, which can be supplied in aninjectable form.

In another aspect the present invention provides methods ofadministering florfenicol to a mammal. The methods involve administeringa composition containing a prodrug of florfenicol to the mammal. In oneembodiment the composition is administered to the mammal by injection.The composition can form a drug depot in the mammal. The prodrug isconverted in vivo by one or more endogenous enzymes into florfenicol. Inone embodiment the prodrug is present in the composition at aconcentration of at least 250 mg/ml. The prodrug is any of the compoundprodrugs described herein, or a combination thereof. In one embodimentthe endogenous enzymes are esterases. In various embodiments the mammalis a bovine, an equine, a porcine, an ovine, a canine, or a feline. Thecompositions can be administered orally, or injected sub-cutaneously,intra-peritoneally, intra-dermally, mucosally, intra-muscularly, or byother means.

A “drug depot” is a reservoir of prodrug situated in the body of themammal and established by the injection of prodrug through the skin. Thedrug depot may have a solid, semi-solid, or liquid form, and representsa concentration of prodrug in the treated mammal. In one embodiment thedrug depot is a precipitate of the prodrug. The depot then becomes asource of prodrug, and therefore active drug, for the treated mammal.The drug depot can be created and situated in a muscle of the mammal byintramuscular injection, but can also be situated in other tissue typessuch as in the dermal layer or in connective tissue. In one embodimentthe depot emits prodrug and the prodrug is converted into active drug inthe body. In another embodiment the prodrug is converted into activedrug at or near the drug depot. In another embodiment the composition inits entirety acts as a drug depot.

By “endogenous enzymes” is meant those enzymes produced or synthesizedwithin the organism by the organism's own protein synthesis apparatus.Endogenous enzymes are those normally found within the organism. Theymay be produced by the mammalian body continuously or produced inresponse to a particular stimulus. “Esterase” is a generic term forthose enzymes that catalyze the hydrolysis of ester bonds. By“intra-muscular” is meant within a muscle. Thus, administration byintra-muscular injection means injected into a muscle.

In another aspect the present invention provides methods ofadministering florfenicol to a mammal. The methods involve injectinginto the mammal a composition comprising one or more compounds of thepresent invention, wherein the one or more compounds is/are convertedinto florfenicol in vivo by the action of one or more endogenousesterases. In various embodiments the one or more compounds is/arepresent in the composition at a concentration of at least 250 mg/ml.

The summary of the invention described above is not limiting and otherfeatures and advantages of the invention will be apparent from thefollowing detailed description, as well as from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 provides a schematic illustration of the prodrug conversionprocess.

FIG. 2 provides a schematic illustration of the synthesis of florfenicolester prodrugs.

FIG. 3 provides a graphical illustration of the in-vitro releasekinetics of florfenicol and florfenicol ester prodrugs.

FIG. 4 provides a graphical illustration of the pharmacokinetics offlorfenicol and florfenicol ester prodrugs in cattle.

FIG. 5 provides a graphical illustration of the pharmacokinetics offlorfenicol butyrate in cats.

FIG. 6 provides a graphical illustration of the pharmacokinetics offlorfenicol hexanoate in cats.

FIG. 7 provides a graphical illustration of the pharmacokinetics offlorfenicol butyrate and hexanoate in cats.

FIG. 8 provides a graphical illustration of serum concentrations offlorfenicol butyrate v. NUFLOR® (Schering-Plough Corp., Kenilworth,N.J.) in cats.

FIGS. 9 a-n provide illustrations of examples of compounds of thepresent invention.

DETAILED DESCRIPTION OF THE INVENTION

The compositions and methods of the present invention have distinct andsurprising advantages over previously available compositions andmethods. While the solubilization of florfenicol usually requiresrelatively strong and harsh solvents, the prodrugs described herein arehighly soluble in mild pharmacologically acceptable solvents, such asglycerol formal.

The prodrug compositions of the present invention are efficientlyconverted to active florfenicol by esterases endogenous in the mammaliansystem. The butyrate and hexanoate prodrug derivatives are less solublein water than free florfenicol and display slower release kinetics,while the acetate prodrug derivative is more soluble in water anddisplays a shorter release time. These chemical characteristics are usedto advantage in the present invention. In general the release rate isrelated to the solubility of the drug in water or biological fluids.When solubility is low, a more viscous depot is formed that releasesprodrug more slowly. When solubility is high, the prodrug is releasedmore quickly from the depot resulting in a faster release rate.Florfenicol can be made more hydrophobic by derivatization into variousesters. In general, the more hydrophobic the derivative the lower thesolubility. Thus, in general the hexanoate derivative is less solublethan the butyrate derivative, which is less soluble than nativeflorfenicol. Thus, when a “fast kill” or “initial burst” of drug isdesired, a more soluble prodrug is advantageous to obtain a shorterrelease time. Longer release times offer the benefit of preventingresidual organisms from growing.

As further described below, a single intramuscular injection of aflorfenicol butyrate prodrug in cattle provided active florfenicol inthe serum at higher levels and for longer periods of time thanintramuscular injections of NUFLOR® (Schering-Plough Corp., Kenilworth,N.J.), which is a commercially available form of florfenicol.Furthermore, a single subcutaneous injection of the butyrate andhexanoate ester prodrug formulations in cats was found to provide serumflorfenicol concentrations above the minimum inhibitory concentration(MIC) for a period of 4-6 days, while serum concentrations of NUFLOR®drop off significantly after 2 days (FIG. 4).

Without being bound by any particular theory, it is believed that afterinjection the water-soluble pharmaceutically acceptable carrier diffusesaway from the injection site, thereby resulting in the formation of adrug depot containing the prodrug. In one embodiment the drug depot is aprecipitate of the prodrug. As the pro-drug slowly dissolves and isreleased from the depot into the serum over time, it is hydrolyzed byendogenous esterases into active florfenicol, thereby providing acontinuous source of florfenicol at effective levels. Thus, thedifficulties associated with florfenicol are overcome in the presentinvention by converting the relatively hydrophilic florfenicol moleculeinto a highly hydrophobic molecule that forms in vivo drug depotsproviding long release times.

Further information regarding the compositions and methods of thepresent invention is provided with reference to the followingnon-limiting examples.

Example 1 Ester Formation

Esters can be formed by the reaction of acids with alcohols. Theformation of esters can be viewed as the substitution of the OH group onthe acid by the alcohol group, and the proton on the alcohol combinedwith the acid OH to form water.

In one embodiment of the invention, the ester is formed by the reactionof an acid anhydride with an alcohol (florfenicol). Thus, an acidanhydride of the derivative(s) being formed is obtained and reactedunder catalytic conditions with florfenicol to form the esterderivative. The catalytic conditions are provided by the presence of astrong acid, such as perchloric or concentrated sulfuric acid. Thereaction is rapid, irreversible, and lies far to the right. Yields ofnearly 100% are obtainable using little or no excess acid anhydride. Thereaction can be depicted as:

In another embodiment florfenicol can be reacted with a carboxylic acidin the presence of a catalyst. This reaction principle is known as“Fischer esterification.” Appropriate catalysts include strong acidssuch as concentrated sulfuric acid, hydrogen chloride, orp-toluenesulfonic acid. This type of reaction is an equilibriumreaction, which must be refluxed for several hours. Higher yields can beobtained by using excessive amounts of acid or alcohol, as the reactiondoes not lie far to the right.

In a third method, esters can be formed by reacting an acid chloridewith florfenicol. The reaction is quantitative and rapid. The acidchloride is the corresponding derivative to be synthesized. Thisreaction produces hydrogen chloride and pyridine can be added toneutralize this reaction product.

The above three methods of forming esters are provided only as examples.Other methods are known to those of ordinary skill in the art, and moremethods may yet be discovered, that can be applied in the presentinvention. Such other methods are also contemplated for use herein.Since esters can be formed from a wide variety of acids, a very largenumber of esters is possible. In the methods, combinations of severalacids can be combined with florfenicol to form a mixture of severaldifferent ester derivatives.

Example 2 Preparation of Prodrugs

Florfenicol acetate, florfenicol butyrate, and florfenicol hexanoateester pro-drugs were synthesized as shown in FIG. 2, using a simple onestep synthetic procedure. The procedure utilized was the acid anhydrideprocedure.

Thus, florfenicol was suspended in the corresponding acid anhydridefollowed by the addition of a catalytic amount of perchloric acid underconditions suitable for esterization to occur such as, for example,stirring at room temperature for about 3 hours. Generally, thisembodiment involves adding solid florfenicol to liquid anhydride. Theflorfenicol is not soluble in anhydride and stays as a suspension. Onceperchloric acid is added, product begins to form and dissolve in thesolution. After sufficient time a completely clear solution is obtainedand product begins to crystallize out of solution. The followingexamples are suitable procedures for preparing prodrugs.

Florfenicol butyrate: 225 g of florfenicol was suspended in 330 ml ofbutyric anhydride in one liter round bottom flask equipped with a overhead stirrer and a drying tube. To this suspension was added one ml ofperchloric acid, while stirring vigorously, with a pipette. A clearsolution was obtained in a few minutes. Stirring was continued foranother 3 hours during which the florfenicol butyrate pro-drug wascrystallized. The solid product was filtered using a Buckner funnelunder vacuum, and washed thoroughly with hexane. The product was finallycrystallized in ethanol and the crystallized product was washed withhexane and dried to obtain around 230 g of solid florfenicol butyrate.

Florfenicol acetate: 250 g of florfenicol was suspended in 330 ml ofacetic anhydride in one liter round bottom flask equipped with aover-head stirrer and a drying tube. To this suspension was added one mlof perchloric acid, while stirring vigorously with a pipette. A clearyellow solution was obtained in a few minutes. Stirring was continuedfor another 3 hours. After 3 hrs, the reaction mixture was poured into1000 g of ice and stirring continued to arrive at a suspension ofproduct. Solid present was filtered and resuspended in 500 ml of water,and then stirred for 10 min to remove residual acetic acid. The solidproduct was filtered using a Buckner funnel under vacuum and thenrecrystallized in ethanol. The recrystallized product was washed withhexane and dried to obtain around 260 g of solid florfenicol acetate.

Florfenicol hexanoate: 100 g of florfenicol was suspended in 500 g ofhexanoic anhydride in one liter round bottom flask equipped with anoverhead stirrer and a drying tube. To this suspension was added 3 ml ofperchloric acid, while stirring vigorously, with a pipette. A clearsolution was obtained in a few minutes. Stirring was continued foranother 3 hours during which the product, florfenicol hexanoate pro-drugwas recrystallized. The solid product was filtered using Buckner funnelunder vacuum, washed thoroughly with hexane. The product was finallyrecrystallized in ethanol and the recrystallized product was washed withhexane and dried to obtain around 117 g of solid florfenicol hexanoate.

Florfenicol phthalate: 3.9 g of florfenicol, 3.6 g of phthalic anhydrideand 7.2 ml of triethyl amine in 36 ml of dioxane were taken in a 100 mlround bottom flask equipped with a magnetic stir bar and a drying tube.The reaction mixture was stirred at room temperature for 6 hours. After6 hours, the contents were poured into 300 ml of cold water. Theresulting gummy solid was dissolved in dichloromethane and washedthoroughly with saturated bicarbonate several times followed by water.The organic layer was dried over anhydrous sodium sulfate andconcentrated over roto-evaporator. The semisolid obtained wasrecrystallized in a mixture of ethylacetate and hexane to obtain a solidproduct of 4.2 grams.

Example 3 In Vitro Release Kinetics

The following example illustrates the preparation of formulations andanalysis of the release kinetics for florfenicol acetate, florfenicolbutyrate, and florfenicol hexanoate. Other prodrug formulations can beprepared and analyzed using the same principles such as, for example,florfenicol propionate, florfenicol pentanoate, florfenicol heptanoate,florfenicol octanoate, florfenicol nanoate, florfenicol decanoate,florfenicol undecanoate, florfenicol dodecanoate, and florfenicolphthalate. Each prodrug was prepared as an injectable formulation at 300mg/ml in non-aqueous carriers including propylene glycol, glycerolformal, N-methyl-pyrrolidone (NMP), and polyethylene glycol (PEG).

7.5 grams of florfenicol butyrate, florfenicol hexanoate, andflorfenicol acetate were transferred to individual 25 ml volumetricflasks. 2.5 ml of propylene glycol was added to each solid, followed bythe addition of glycerol formal to a final volume of 25 ml. The flaskwas placed on a shaker for about 2 hours to obtain a clear homogeneoussolution.

In vitro release kinetics of the prodrug formulations and a solution offlorfenicol in NMP were studied using a dialysis technique. A 0.5 mlaliquot of each formulation was injected into a SLIDE-A-LYZER® dialysiscassette (Pierce Biotechnology, Inc., Rockford, Ill.), and each cassettewas suspended in a flask containing 150 ml of phosphate-buffered saline,10 mM phosphate, 120 mM NaCl, pH 7.4.

The ester prodrug was observed to precipitate in the bag within about anhour. The acetate prodrug was found to be more soluble in aqueouscarriers, while the butyrate and hexanoate derivatives formedprecipitates more readily and were less soluble in aqueous carriers suchas the phosphate buffer. Aliquots were removed from the flasks atvarious intervals and florfenicol esters were determined by HPLC. The invitro release samples were directly injected after filtering through a0.2 um filter in phosphate-buffered saline.

Samples were analyzed on a reversed phase C-18 column with detection at223 nm, using acetonitrile and water as the solvent system. The gradientstarted at 25% acetonitrile rising to 60% acetonitrile over 10 minutes.From 20-42 minutes the acetonitrile was lowered to 25%. The flow ratewas 1.2 ml/min.

FIG. 3 illustrates the release kinetics for each florfenicol prodrug.The acetate prodrug was found to display faster release kineticscompared to florfenicol, while the butyrate and hexanoate derivativesdisplayed slower release kinetics compared to native florfenicol.

Example 4 Minimum Inhibitory Concentrations

The Minimum Inhibitory Concentrations (MICs) were determined for eachester derivative in the presence and absence of pig liver esterase (PLE)with florfenicol (NUFLOR®, Schering-Plough, Kenilworth, N.J.) as acontrol. The results are summarized in Table 1. The results illustratethat the prodrugs have comparable MICs to the native florfenicol in thepresence of PLE, while no activity was observed in the absence of theenzyme. Higher MICs were observed with the acetate prodrug than with thebutyrate and hexanoate prodrugs, presumably because acetate is a poorsubstrate for PLE.

TABLE 1 Determination of MICs for Florfenicol Derivatives MIC (ug/ml)Nuflor ® FFAc FFBut FFHex. Nuflor ® FFAc FFBut FFHex. w/est. w/est.w/est. w/est. E. coli 25922 6.25 >200 >200 >200 6.25 200 25 12.5Klebsiella pneumoniae 31488 1.56 200 >200 >200 1.56 100 12.5 6.25 Pseud.aeruginosa 27853 200 >200 >200 >200 >200 >200 >200 >200 Serratiamarcescens 14757 25 >200 >200 >200 25 200 100 25 Proteus mirabilis 299066.25 200 >200 >200 6.25 200 25 6.25 Dilution series: 200, 100, 50, 25,12.5, 6.25, 3.125, 1.56 Key FFAc = Florfenicol acetate FFBut =Florfenicol butyrate FFHex = Florfenicol hexanoate w/est. = withesterase

Example 5 Hydrolysis of Prodrug by Esterases

The conversion of florfenicol esters to florfenicol by the esterasespresent in dog and cat serum and whole blood was studied. Each of theprodrugs to be analyzed was prepared at a concentration of 300 mg/mlsolution of the prodrug in 10% propylene glycol in glycerol formal. 0.1ml aliquots of these preparations were spiked into 1.933 ml of blood andserum and the conversion to active florfenicol at 37° C. was studied.100 ul aliquots were loaded onto a solid phase extraction (SPE)cartridge and the drug/pro-drug was extracted. The results aresummarized in Table 2 and illustrate that all three esters aresubstrates to both canine and feline esterases.

TABLE 2 Percent Hydrolysis of Florfenicol Esters in Canine Plasma andWhole Blood After 24 Hrs Plasma Whole blood Florfenicol 100 100Florfenicol acetate 51 96 Florfenicol butyrate 80 96 Florfenicolhexanoate 94 88

Example 6 Animal to Animal Variations

Animal to animal variations in hydrolysis rates were also examined.Florfenicol butyrate was spiked into whole blood from five differentdogs and the percent conversion to florfenicol after 30 minutes wasdetermined by HPLC analysis. The results are shown in Table 3 andillustrate that no significant change in the hydrolysis rate wasobserved within the group of five animals.

TABLE 3 Percent Hydrolysis of Florfenicol Butyrate in Canine Whole BloodAfter 30 Minutes Dog# % Hydrolysis 34410 83 35752 94 35803 98 35870 9035872 100

Example 7 Intra-Muscular Administration to Cattle

This example describes the administration of various florfenicol esterprodrug formulations by injection into cattle with the serum beinganalyzed for the presence of florfenicol and prodrug.

HPLC analysis of the serum from the injected animals showed aflorfenicol peak and no ester derivative peaks. These results illustratethe conversion of prodrug to active florfenicol in vivo.

Florfenicol butyrate was formulated at 350 mg/ml in 10% propylene glycolin glycerol formal as described above and injected intramuscularly intofour calves at a dosage of 40 mg/kg calf. Commercially available NUFLOR®was used as a control and was injected as a single dose of 20 mg/kg asper label. Injections were administered to the muscles of the neck orthe buttocks. Serum samples from the injected animals were analyzed forflorfenicol and the results are summarized in FIG. 4. With NUFLOR®, theserum concentrations built up rapidly to a high of about 4.5 mg/ml, andthen declined rapidly. With the florfenicol butyrate the serumconcentrations also built up rapidly but decreased more slowly, thusextending the area under curve and retaining active florfenicol in theserum at effective levels for a longer period of time. Thus, during days2-7 the serum concentrations obtained using florfenicol butyrate werehigher compared to those where NUFLOR® was used. These data illustratethat the pro-drug formulation provided for the controlled release offlorfenicol, thus keeping therapeutic levels of florfenicol in the serumfor a longer period time, while the NUFLOR® formulation resulted in arapid build up and a rapid decrease.

Example 8 Sub-Cutaneous Florfenicol Butyrate in Cats

Florfenicol butyrate was formulated at 300 mg/ml in 10% propylene glycolin glycerol formal as described above and injected sub cutaneously intotwo cats at a dose of 40 mg/kg of cat. Both animals showed serumconcentrations of florfenicol of greater than 1.0 ug/ml (the MIC) for5-6 days, with serum concentrations peaking at 4-5 ug/ml. Anillustration of the data is provided in FIG. 5.

In a separate experiment florfenicol butyrate was formulated at 350mg/ml in 10% propylene glycol in glycerol formal and injectedsubcutaneously into five cats at a dosage of 40 mg/kg. Commerciallyavailable NUFLOR® was used as a control and was also injected at adosage of 40 mg/kg. Serum samples from the injected animals wereanalyzed for the presence of florfenicol, and the results aregraphically depicted in FIG. 8. Concentrations of NUFLOR® built uprapidly in the serum and then declined rapidly over two days.Conversely, the concentration of florfenicol butyrate built up lessrapidly and declined more slowly than that of NUFLOR®. Thus, theflorfenicol butyrate was released more slowly than NUFLOR®. FIG. 8demonstrates that serum concentrations of florfenicol butyrate arehigher compared to NUFLOR® for days 2-6. Therefore, the florfenicolpro-drug formulation provided the controlled release of the active attherapeutic levels for a longer period of time than NUFLOR®.

Example 9 Sub-Cutaneous Florfenicol Hexanoate in Cats

In this example florfenicol hexanoate was formulated at 300 mg/ml in 10%propylene glycol in glycerol formal as described above and injectedsub-cutaneously into two cats at a dose of 40 mg/kg of cat. Both animalsshowed serum florfenicol concentrations above 1.0 ug/ml for 6 days withhigh concentrations at 2.5-4 ug/ml. The results are illustrated in FIG.6.

Example 10 Mixture of Florfenicol Derivatives Sub-Cutaneous in Cats

In this example a 1:1 ratio mixture of florfenicol butyrate andflorfenicol hexanoate was formulated at 300 mg/ml in 10% propyleneglycol in glycerol formal as described above and injectedsub-cutaneously into two cats at a dose of 40 mg/kg of cat. Both animalsshowed serum concentrations of florfenicol of greater than 1.0 ug/ml for4-6 days, with high concentrations of 2.5-10 ug/ml. The results aregraphically illustrated in FIG. 7.

Example 11 Florfenicol Acetate Sub-Cutaneous in Cats

In this example florfenicol acetate was formulated at 300 mg/ml in 10%propylene glycol in glycerol formal as described above and injectedsub-cutaneously into a cat at a dose of 40 mg/kg of cat. Serum sampleswere analyzed periodically at 0, 1, 6, and 24 hours after the injection.The results are summarized below in Table 4, which indicates the serumconcentrations of florfenicol and acetate at the indicated timeintervals after injection. A considerably higher concentration of drugwas found at 24 hours after injection than found when using the butyrateand hexanoate derivatives. Un-hydrolyzed acetate ester was also noted ata serum concentration of about 5 ug/ml.

Without being bound by any particular theory, it is believed by theinventors that the acetate esters are released more rapidly into thebloodstream than the butyrate and hexanoate esters. However, thehydrolysis rate is less with the acetate ester, which may be caused byit being a poorer substrate for the endogenous esterases.

Time (hrs) 0 hours 1 hour 6 hours 24 hours [Florfenicol] (ug/ml) 0 4.1815.49 3.93 [Acetate] (ug/ml) 0 5.70 5.4 0.5

The invention illustratively described herein may be practiced in theabsence of any element or elements, limitation or limitations that arenot specifically disclosed herein. The terms and expressions which havebeen employed are used as terms of description and not of limitation,and there is no intention that in the use of such terms and expressionsof excluding any equivalents of the features shown and described orportions thereof, but it is recognized that various modifications arepossible within the scope of the invention claimed. Thus, it should beunderstood that although the present invention has been specificallydisclosed by various embodiments and optional features, modification andvariation of the concepts herein disclosed may be resorted to by thoseskilled in the art, and that such modifications and variations areconsidered to be within the scope of this invention as defined by theappended claims.

The contents of the articles, patents, and patent applications, and allother documents and electronically available information mentioned orcited herein, are hereby incorporated by reference in their entirety tothe same extent as if each individual publication was specifically andindividually indicated to be incorporated by reference. Applicantsreserve the right to physically incorporate into this application anyand all materials and information from any such articles, patents,patent applications, or other documents.

1. A method of treating a bacterial infection in a bovine comprising administering to the bovine a composition comprising a florfenicol ester and a pharmaceutically acceptable solvent wherein the florfenicol ester is selected from the group consisting of florfenicol acetate, florfenicol propionate, florfenicol butyrate, florfenicol pentanoate, florfenicol heptanoate, florfenicol octanoate, florfenicol nanoate, florfenicol decanoate, florfenicol undecanoate, and florfenicol dodecanoate; the bacterial infection is susceptible to florfenicol; the composition is administered as a single injection in an amount sufficient to treat the infection without requiring a second injection; and wherein administering a 40 mg/kg dose of the florfenicol ester provides a Cmax for the concentration of florfenicol in the bovine that is lower than the Cmax that would be obtained if the bovine was administered a comparative composition at a dose of 20 mg/kg, wherein the comparative composition contains 300 mg of florfenicol, 250 mg N-methyl-2-pyrrolidone, 150 mg propylene glycol, and sufficient polyethylene glycol to provide 1 mL of the comparative composition.
 2. The method of claim 1, wherein the administering is selected from the group consisting of intra-muscular injection, intra-venous injection, sub-cutaneous injection, intra-dermal injection, intra-peritoneal injection, and parenteral injection.
 3. The method of claim 2, wherein the administering is intra-muscular injection.
 4. The method of claim 1, wherein the concentration of the florfenicol ester in the pharmaceutically acceptable organic solvent is at least 200 mg/mL.
 5. The method of claim 1, wherein the pharmaceutically acceptable solvent is selected from the group consisting of pyrrolidone, N-methyl pyrrolidone, polyethylene glycol, propylene glycol, glycerol formal, isosorbid dimethyl ether, ethanol, dimethyl sulfoxide, tetrahydrofurfuryl alcohol, triacetin, or a combination thereof.
 6. The method of claim 1, wherein the pharmaceutically acceptable solvent is about 10% propylene glycol in glycerol formal.
 7. The method of claim 6, wherein the florfenicol ester is florfenicol butyrate.
 8. The method of claim 1, wherein the florfenicol ester is florfenicol butyrate.
 9. The method of claim 1, wherein the bacterial infection is caused by a bacteria from the group consisting of Citrobacter, Proteus mirabilis, Proteus sp., Shigella, Salmonella, Providencia, Bacteroides, Staphylococcos aureus, Enterococci, Pasteurella haemolytica, Pasteurella multocida, Haemophilus somnus, and Haemophilus influenza.
 10. The method of claim 1, wherein the administering provides a serum concentration of florfenicol in the bovine that is higher two days after administration than the serum concentration that would be obtained if the bovine was administered a single injection of the comparative composition at a dose of 20 mg/kg.
 11. The method of claim 10, wherein the administering provides a serum concentration of florfenicol in the bovine that is higher three days after administration than the serum concentration that would be obtained if the bovine was administered the comparative composition at a dose of 20 mg/kg.
 12. The method of claim 1, wherein the composition is administered at a dose of about 40 mg/kg.
 13. The method of claim 1, wherein the administering provides a drug depot in the bovine.
 14. The method of claim 1, wherein the bacterial infection is a respiratory infection.
 15. The method of claim 1, wherein the florfenicol ester is florfenicol butyrate, the administering is intra-muscular injection, the administering provides a serum concentration of florfenicol in the bovine that is higher three days after administration than the serum concentration that would be obtained if the bovine was administered a single injection of the comparative composition at a dose of 20 mg/kg.
 16. The method of claim 15, wherein the composition is administered at a dose of about 40 mg/kg. 